Journal: European journal of cell biology

Article Title: Receptor-mediated internalization promotes increased endosome size and number in a RAB4- and RAB5-dependent manner

doi: 10.1016/j.ejcb.2023.151339

Figure Lengend Snippet: EE/SE size and number increase upon epidermal growth factor internalization. Non-small cell lung cancer cells were grown on cover-slips and either mock-treated or incubated with 2 ug/ml epidermal growth factor-555 (EGF-555) for 15 min. Cells were then fixed in 4% paraformaldehyde and subjected to immunostaining with antibodies to the EE/SE marker protein, RAB5. 4–5 images derived from 5 serial z-sections were obtained from 3 independent experiments and mean endosomal area of RAB5 structures (A) or number of RAB5-containing endosomes (B) were measured for cells incubated with or without EGF using Imaris software. (C) Representative image and inset for cells with no EGF uptake. (D) Representative image and inset for cells with EGF uptake. The p-value for EE/SE size was calculated with a Mann-Whitney test, because the data did not meet the assumption of normality by the D’Agostino Pearson test. The p-value for EE/SE number was calculated with an unpaired two-tailed t-test, because the data met the assumption for normality using the D’Agostino Pearson test.

Article Snippet: Primary and secondary antibodies used in this study were rabbit anti-EEA1 (Cell Signaling cat. no. C45810), mouse anti-RAB5 (BD Transduction Labs cat. no. 610724), mouse anti-RAB4 (BD Transduction Labs cat. no. 610889), rabbit anti-RAB8 (Abcam cat no. 237702), rabbit anti-RAB10 (Abcam cat. no. 237703), mouse anti-RAB11 (BD Transduction Labs cat. no. 610657), HRP-conjugated anti-vinculin (Cell Signaling cat no. E1E9V XP), goat anti-mouse horseradish peroxidase (HRP), (cat. no. 115–035–003; Jackson ImmunoResearch Laboratories, West Grove, PA), donkey anti-rabbit HRP (cat. no. NA934V; GE Healthcare, Pittsburgh, PA), Alexa 568–conjugated goat anti-mouse (cat. no. A11031), and Alexa 488–conjugated goat anti-rabbit (cat. no. A11034; Life Technologies, Carlsbad, CA).

Techniques: Incubation, Immunostaining, Marker, Derivative Assay, Software, MANN-WHITNEY, Two Tailed Test

Journal: European journal of cell biology

Article Title: Receptor-mediated internalization promotes increased endosome size and number in a RAB4- and RAB5-dependent manner

doi: 10.1016/j.ejcb.2023.151339

Figure Lengend Snippet: The number of EE/SE per cell is regulated by select endosomal RAB proteins. (A) HeLa cells were grown on cover-slips and 6-well plates and either mocktreated (transfection reagent only) or incubated with the following amounts of siRNA oligonucleotides specific for RAB4, RAB5, RAB8A, RAB10 or RAB11A per 2 ml well for 48 h: RAB4, 280 nmol; RAB5, 400 nmol; RAB8A, 600 nmol, RAB10, 400 nmol; RAB11A, 600 nmol. Cells from the 6-well plates were collected, lysed and subjected to immunoblot analysis with antibodies to RAB4, RAB5, RAB8A, RAB10 and RAB11 (to validate knock-down efficacy), and to vinculin (loading control). The solid line separating mock and RAB11A from the other RAB proteins indicates that this pair of lanes was run on a separate gel due to space constraints. Gels and images are representative of 3 independent experiments. (B-G) Representative images of cells immunostained for EEA1 upon mock-treatment (B), RAB4 siRNA-treatment (C), RAB5 siRNA-treatment (D), RAB8A siRNA-treatment (E), RAB10 siRNA-treatment (F), RAB11A siRNA-treatment (G). (H) Graph illustrating the number of EE/SE observed per field of mock-treated and siRNA knock-down cells, with or without 15 min transferrin uptake. Data provided is a mean and standard deviation from 3 independent experiments. 3–5 images were obtained from each treatment, and Imaris software was used to measure EE/SE numbers. The p-values for EE/SE numbers were calculated with a Mann-Whitney test, because the data did not meet the assumption of normality by the D’Agostino Pearson or Kolmogorov-Smirnov tests. (I) Graph depicting the area of EEA1 endosomes observed per field of mock-treated and siRNA knock-down cells, with or without 15 min transferrin uptake. Data provided are mean and standard deviations from 3 independent experiments. 3–5 images were obtained from each treatment, and Imaris software was used to measure endosome area. The p-values for EEA1 endosome areas were calculated by t-test, as the data met the assumption of normality by the Kolmogorov-Smirnov test.

Article Snippet: Primary and secondary antibodies used in this study were rabbit anti-EEA1 (Cell Signaling cat. no. C45810), mouse anti-RAB5 (BD Transduction Labs cat. no. 610724), mouse anti-RAB4 (BD Transduction Labs cat. no. 610889), rabbit anti-RAB8 (Abcam cat no. 237702), rabbit anti-RAB10 (Abcam cat. no. 237703), mouse anti-RAB11 (BD Transduction Labs cat. no. 610657), HRP-conjugated anti-vinculin (Cell Signaling cat no. E1E9V XP), goat anti-mouse horseradish peroxidase (HRP), (cat. no. 115–035–003; Jackson ImmunoResearch Laboratories, West Grove, PA), donkey anti-rabbit HRP (cat. no. NA934V; GE Healthcare, Pittsburgh, PA), Alexa 568–conjugated goat anti-mouse (cat. no. A11031), and Alexa 488–conjugated goat anti-rabbit (cat. no. A11034; Life Technologies, Carlsbad, CA).

Techniques: Transfection, Incubation, Western Blot, Standard Deviation, Software, MANN-WHITNEY

Journal: European journal of cell biology

Article Title: Receptor-mediated internalization promotes increased endosome size and number in a RAB4- and RAB5-dependent manner

doi: 10.1016/j.ejcb.2023.151339

Figure Lengend Snippet:

Article Snippet: Primary and secondary antibodies used in this study were rabbit anti-EEA1 (Cell Signaling cat. no. C45810), mouse anti-RAB5 (BD Transduction Labs cat. no. 610724), mouse anti-RAB4 (BD Transduction Labs cat. no. 610889), rabbit anti-RAB8 (Abcam cat no. 237702), rabbit anti-RAB10 (Abcam cat. no. 237703), mouse anti-RAB11 (BD Transduction Labs cat. no. 610657), HRP-conjugated anti-vinculin (Cell Signaling cat no. E1E9V XP), goat anti-mouse horseradish peroxidase (HRP), (cat. no. 115–035–003; Jackson ImmunoResearch Laboratories, West Grove, PA), donkey anti-rabbit HRP (cat. no. NA934V; GE Healthcare, Pittsburgh, PA), Alexa 568–conjugated goat anti-mouse (cat. no. A11031), and Alexa 488–conjugated goat anti-rabbit (cat. no. A11034; Life Technologies, Carlsbad, CA).

Techniques: Sequencing, Concentration Assay

Journal: bioRxiv

Article Title: TBCK-deficiency leads to compartment-specific mRNA and lysosomal trafficking defects in patient-derived neurons

doi: 10.1101/2025.03.02.641041

Figure Lengend Snippet: a, Representative confocal images showing RAB5 and ( b ) EEA1 in control (Ctrl) TBCK-deficient neurons (p.R126X). Inset magnifications (orange squares) show the distribution of early endosomes. c, Confocal images show PPP1R21 and RAB5 in control (Ctrl) TBCK-deficient neurons (p.R126X). Different inset magnifications (orange and blue squares) show the distribution and colocalization of PPP1R21 and RAB5. d, Confocal images showing PPP1R21 and EEA1 in control (Ctrl) TBCK-deficient neurons (p.R126X). Different inset magnifications (orange and blue squares) show the distribution and colocalization of PPP1R21 and EEA1. e, Representative immunoblot and ( f ) quantification of the early endosome marker, RAB5, in control (Ctrl) TBCK-deficient neurons (p.R126X). Significance was calculated using unpaired t-test analysis. g, Analysis of endosome size showing larger endosomes in TBCK-deficient neurons (p.R126X) compared to controls (Ctrl) (n=3). Significance was calculated using unpaired t-test analysis. h, Quantification of endosomes number shows fewer number of endosomes (EEA1+) in TBCK-deficient neurons (p.R126X) compared to controls (Ctrl) (n=3). Significance was calculated using unpaired t-test analysis. i, EEA1 and ( j ) PPP1R21 distribution along compartments of control (Ctrl) TBCK-deficient neurons (p.R126X). Cell compartments were identified and traced using Fiji, ImageJ (n=3). Significance was calculated using unpaired t-test analysis. k, Confocal images show PPP1R21 and EEA1 in control (Ctrl) TBCK-deficient fibroblasts (p.R126X). Different inset magnifications (orange squares) show the distribution and colocalization of PPP1R21 and EEA1. l, Staining of PPP1R21 and EEA1 in HEK293 cells transduced with ShRNA scramble (Sh Scr) and ShRNA TBCK#2 (Sh TBCK). Different inset magnifications (orange squares) show the distribution and colocalization of PPP1R21 and EEA1. *p<0.01, **p<0.05 and ***p<0.001. All graphs show error bars with mean ± SD from independent experiments (n).

Article Snippet: The following antibodies were used: rabbit anti-LC3B (1:5000, Cell signaling, 27755), mouse anti-p62 (1:500, Abcam, ab56416), mouse anti-RAB5 (1:500, Cell signaling, 2157), chicken anti-TUJ1 (1:1000, Aves labs), mouse anti-Flag (1:1000, Sigma, F1804), rabbit anti-TBCK (1:1000, Novus, NBP1-83166), mouse anti-LAMP1 (1:250, DSHB, H4A3), goat anti-LAMP2 (1:250, R&D Systems, AF6228), rabbit anti-PPP1R21 (1:500, Atlas Ab, HPA036791), rabbit anti-C12orf4 (1:500, Sigma, HPA037871), rabbit anti-JIP4 (1:500, Cell signaling, 5519), rabbit anti-TRIM27 (1:500, IBL, 18791), mouse anti-EEA1 (1:500, BD Tranduction), goat anti-mouse IgG Alexa Fluor 488 (1:500, ThermoFisher, A-11001), goat anti-mouse IgG Alexa Fluor 555 (1:500, ThermoFisher, A-21422), goat anti-mouse IgG Alexa Fluor 647 (1:500, ThermoFisher, A-21235), goat anti-rabbit IgG Alexa Fluor 488 (1:500, ThermoFisher, A-11008), goat anti-rabbit IgG Alexa Fluor 555 (1:500, ThermoFisher, A-21428), goat anti-rabbit IgG Alexa Fluor 647 (1:500, ThermoFisher, A-21245), donkey anti-goat IgG Alexa Fluor 488 (1:500, ThermoFisher, A-11055), donkey anti-goat IgG Alexa Fluor 555 (1:500, ThermoFisher, A-21432), donkey anti-goat IgG Alexa Fluor 647 (1:500, ThermoFisher, A-21447), goat anti-chicken IgG Alexa Fluor 488 (1:500, ThermoFisher, A-11039), goat anti-chicken IgG Alexa Fluor 555 (1:500, ThermoFisher, A-21437), goat anti-chicken IgG Alexa Fluor 647 (1:500, ThermoFisher, A-21449).

Techniques: Control, Western Blot, Marker, Staining, Transduction, shRNA

Journal: bioRxiv

Article Title: TBCK-deficiency leads to compartment-specific mRNA and lysosomal trafficking defects in patient-derived neurons

doi: 10.1101/2025.03.02.641041

Figure Lengend Snippet: a, Representative images showing the distribution of PPP1R21 with RAB5 or ( b ) EEA1 in control (Ctrl) TBCK-deficient neurons (p.R126X). c, HyVolution confocal images show the interaction between PPP1R21 and EEA1 in control (Ctrl) and TBCK-deficient fibroblasts (p.R126X). d, Confocal images show TBCK and EEA1 in control (Ctrl) TBCK-deficient neurons (p.R126X). Different inset magnifications (orange and blue squares) show the distribution and colocalization of TBCK and EEA1.

Article Snippet: The following antibodies were used: rabbit anti-LC3B (1:5000, Cell signaling, 27755), mouse anti-p62 (1:500, Abcam, ab56416), mouse anti-RAB5 (1:500, Cell signaling, 2157), chicken anti-TUJ1 (1:1000, Aves labs), mouse anti-Flag (1:1000, Sigma, F1804), rabbit anti-TBCK (1:1000, Novus, NBP1-83166), mouse anti-LAMP1 (1:250, DSHB, H4A3), goat anti-LAMP2 (1:250, R&D Systems, AF6228), rabbit anti-PPP1R21 (1:500, Atlas Ab, HPA036791), rabbit anti-C12orf4 (1:500, Sigma, HPA037871), rabbit anti-JIP4 (1:500, Cell signaling, 5519), rabbit anti-TRIM27 (1:500, IBL, 18791), mouse anti-EEA1 (1:500, BD Tranduction), goat anti-mouse IgG Alexa Fluor 488 (1:500, ThermoFisher, A-11001), goat anti-mouse IgG Alexa Fluor 555 (1:500, ThermoFisher, A-21422), goat anti-mouse IgG Alexa Fluor 647 (1:500, ThermoFisher, A-21235), goat anti-rabbit IgG Alexa Fluor 488 (1:500, ThermoFisher, A-11008), goat anti-rabbit IgG Alexa Fluor 555 (1:500, ThermoFisher, A-21428), goat anti-rabbit IgG Alexa Fluor 647 (1:500, ThermoFisher, A-21245), donkey anti-goat IgG Alexa Fluor 488 (1:500, ThermoFisher, A-11055), donkey anti-goat IgG Alexa Fluor 555 (1:500, ThermoFisher, A-21432), donkey anti-goat IgG Alexa Fluor 647 (1:500, ThermoFisher, A-21447), goat anti-chicken IgG Alexa Fluor 488 (1:500, ThermoFisher, A-11039), goat anti-chicken IgG Alexa Fluor 555 (1:500, ThermoFisher, A-21437), goat anti-chicken IgG Alexa Fluor 647 (1:500, ThermoFisher, A-21449).

Techniques: Control

Journal: bioRxiv

Article Title: TBCK-deficiency leads to compartment-specific mRNA and lysosomal trafficking defects in patient-derived neurons

doi: 10.1101/2025.03.02.641041

Figure Lengend Snippet: a, Representative confocal images in control (Ctrl) and TBCK-deficient neurons (p.R126X) showing PPP1R21, EEA1 (early endosomes) and LAMP2 (lysosomes). Inset magnifications (orange and blue squares) show the distribution and colocalization of PPP1R21, EEA1 and LAMP2. b, Selected confocal images show PPP1R21, EEA1 and LAMP2 in control (Ctrl) and TBCK-deficient fibroblasts (p.R126X). Different inset magnifications (orange squares) show the distribution and colocalization of PPP1R21, EEA1 and LAMP2. c, Confocal images of HEK293 cells transduced with ShRNA scramble (Sh Scr) and ShRNA TBCK#2 (Sh TBCK). Different inset magnifications (orange squares) show the distribution and colocalization of PPP1R21, EEA1 and LAMP2. d, Colocalization analysis using the Mander’s overlap coefficient (MOC) to quantity the degree of colocalization between PPP1R21 with LAMP2; LAMP2 with EEA1; and PPP1R21 with EEA1 in control and TBCK-deficient fibroblasts (p.R126X) (n=3). e, Function of TBCK in control (normal conditions) and TBCK-deficient cells. In presence of TBCK, one portion of PPP1R21 is localized in early endosomes with RAB5 and EEA1, but also another portion can be observed distributed in the cytoplasm. In absence of TBCK, PPP1R21 in mainly localized into large perinuclear vesicles formed by early endosomal (EEA1) and lysosomal (LAMP1/2) markers. This suggest that the PPP1R21 regulation by TBCK is essential for the formation, distribution or maturation of endosomes and lysosomes. These alterations impact also in the distribution and trafficking of mRNA mediated through the endolysosomal pathway which led to cellular defects and subsequently to TBCK Encephaloneuronopathy. p<0.01, **p<0.05 and ***p<0.001; Student’s t test; all graphs show error bars with mean ± SD from independent experiments (n).

Article Snippet: The following antibodies were used: rabbit anti-LC3B (1:5000, Cell signaling, 27755), mouse anti-p62 (1:500, Abcam, ab56416), mouse anti-RAB5 (1:500, Cell signaling, 2157), chicken anti-TUJ1 (1:1000, Aves labs), mouse anti-Flag (1:1000, Sigma, F1804), rabbit anti-TBCK (1:1000, Novus, NBP1-83166), mouse anti-LAMP1 (1:250, DSHB, H4A3), goat anti-LAMP2 (1:250, R&D Systems, AF6228), rabbit anti-PPP1R21 (1:500, Atlas Ab, HPA036791), rabbit anti-C12orf4 (1:500, Sigma, HPA037871), rabbit anti-JIP4 (1:500, Cell signaling, 5519), rabbit anti-TRIM27 (1:500, IBL, 18791), mouse anti-EEA1 (1:500, BD Tranduction), goat anti-mouse IgG Alexa Fluor 488 (1:500, ThermoFisher, A-11001), goat anti-mouse IgG Alexa Fluor 555 (1:500, ThermoFisher, A-21422), goat anti-mouse IgG Alexa Fluor 647 (1:500, ThermoFisher, A-21235), goat anti-rabbit IgG Alexa Fluor 488 (1:500, ThermoFisher, A-11008), goat anti-rabbit IgG Alexa Fluor 555 (1:500, ThermoFisher, A-21428), goat anti-rabbit IgG Alexa Fluor 647 (1:500, ThermoFisher, A-21245), donkey anti-goat IgG Alexa Fluor 488 (1:500, ThermoFisher, A-11055), donkey anti-goat IgG Alexa Fluor 555 (1:500, ThermoFisher, A-21432), donkey anti-goat IgG Alexa Fluor 647 (1:500, ThermoFisher, A-21447), goat anti-chicken IgG Alexa Fluor 488 (1:500, ThermoFisher, A-11039), goat anti-chicken IgG Alexa Fluor 555 (1:500, ThermoFisher, A-21437), goat anti-chicken IgG Alexa Fluor 647 (1:500, ThermoFisher, A-21449).

Techniques: Control, Transduction, shRNA

Journal: Journal of Cell Science

Article Title: His domain protein tyrosine phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation

doi: 10.1242/jcs.259192

Figure Lengend Snippet: EGFR activated by HB-EGF accumulation in Rab5-positive endosomes is specific to HD-PTP depletion. Control HeLa cells or cells depleted of HD-PTP or Alix were incubated with fluorescently labelled EGFR, stimulated with HB-EGF, chased for up to 180 min and immunostained for Rab5. Arrows indicate colocalised Rab5 and EGFR puncta; arrowheads indicate undegraded EGFR not colocalised with Rab5-positive endosomes. Asterisks indicate nuclei from binucleate cells. Representative data from three independent experiments. The percentages indicate the proportion of EGFR-containing structures that also labelled for Rab5. Scale bar: 10 µm.

Article Snippet: Rabbit: anti-CHMP4B (Proteintech; Cat. 13683-1-AP; batch 00019545; 1:250 for IF); anti-HD-PTP (Proteintech; Cat. 10472-1-AP; batch 00018154; 1:5000 for WB; 1:50 for IF) ( ); anti-UBAP1 (Proteintech; Cat. 12385-1-AP; batch 00014891) ( ); anti-EEA1 (Cell Signaling Technologies; C45B10; Cat. 3288S; batch 8; 1:400 for IF); anti-Rabex-5 (Thermo Scientific - Invitrogen; Cat. PA5-21117; batch RF2221849; 1:2500 for WB); anti-Rabaptin-5 (Proteintech; Cat. 14350-1-AP; batch 00005401; 1:5000 for WB); anti-Rab5 clone C8B1 (Cell Signaling Technologies; Cat. 3547S; batch 7; 1:500 for IF; 1:2500 for WB); anti-GAPVD1 (Abcam; Cat. ab86033; batch GR265707-8; 1:5000 for WB); anti-VPS4B (Proteintech; Cat. 17673-1-AP; batch 00009255; 1:2500 for WB); anti-exportin-1 (Bethyl Laboratories; Cat. A300-469A; batch 2).

Techniques: Incubation

Journal: Journal of Cell Science

Article Title: His domain protein tyrosine phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation

doi: 10.1242/jcs.259192

Figure Lengend Snippet: The effect of HD-PTP depletion on endosomal clustering is not secondary to ESCRT depletion. (A) Endosomal clusters induced by HD-PTP depletion is not an off-target effect. HeLa cells were depleted with siRNA and rescued with RNAi-resistant HA–HD-PTP as indicated and immunostained for Rab5 and EEA1. Graph shows quantification of the approximate area of endosomal structures. Values represent mean±s.d. ( n =10). *** P <0.0001 (one-way ANOVA with Bonferroni's test for multiple comparisons). (B) Validation of siRNA silencing of VPS4B. Values represent mean±s.d. ( n =4). *** P <0.0001 (unpaired two-tailed Student's t -test). (C) Aberrant ESCRT-III accumulation caused by HD-PTP depletion is not responsible for clustering of Rab5 endosomes. HeLa cells were depleted with siRNA as indicated and immunostained for Rab5 and CHMP4B. Representative data from three independent experiments. Scale bars: 20 µm.

Article Snippet: Rabbit: anti-CHMP4B (Proteintech; Cat. 13683-1-AP; batch 00019545; 1:250 for IF); anti-HD-PTP (Proteintech; Cat. 10472-1-AP; batch 00018154; 1:5000 for WB; 1:50 for IF) ( ); anti-UBAP1 (Proteintech; Cat. 12385-1-AP; batch 00014891) ( ); anti-EEA1 (Cell Signaling Technologies; C45B10; Cat. 3288S; batch 8; 1:400 for IF); anti-Rabex-5 (Thermo Scientific - Invitrogen; Cat. PA5-21117; batch RF2221849; 1:2500 for WB); anti-Rabaptin-5 (Proteintech; Cat. 14350-1-AP; batch 00005401; 1:5000 for WB); anti-Rab5 clone C8B1 (Cell Signaling Technologies; Cat. 3547S; batch 7; 1:500 for IF; 1:2500 for WB); anti-GAPVD1 (Abcam; Cat. ab86033; batch GR265707-8; 1:5000 for WB); anti-VPS4B (Proteintech; Cat. 17673-1-AP; batch 00009255; 1:2500 for WB); anti-exportin-1 (Bethyl Laboratories; Cat. A300-469A; batch 2).

Techniques: Two Tailed Test

Journal: Journal of Cell Science

Article Title: His domain protein tyrosine phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation

doi: 10.1242/jcs.259192

Figure Lengend Snippet: Depletion of HD-PTP enhances Rab5 activity on endosomes. (A) HeLa cells were depleted with siRNA as indicated and immunostained for EEA1. Histogram shows the percentage of cells that displayed a normal distribution of EEA1 (mean±s.d. from at least three independent experiments, 100–300 cells counted per experiment, total number of cells indicated above each histogram bar). *** P <0.0001; ns, not significant (one-way ANOVA with Bonferroni's test for multiple comparisons). RBPT5, Rabaptin-5. (B) As A, but immunostained for Rab5. (C) HeLa cells were depleted with siRNA as indicated and transfected with NowGFP–Rab5a and mRuby–EEA1.sh-R5BD. The fluorescence lifetime of NowGFP–Rab5a was measured. Panels show the scale and distribution of fluorescence lifetime in representative cells (lookup chart key on right, black/blue=0 ns, red=7000 ns). Arrowheads indicate pockets of decreased fluorescence lifetime. Graph shows quantification of fluorescence lifetimes over whole cell areas. Values represent mean±s.d. ( n =8–18 cells from three independent experiments, total number of cells indicated above each histogram bar). *** P <0.0001, ns, not significant (one-way ANOVA with Bonferroni's test for multiple comparisons). Scale bars: 20 µm.

Article Snippet: Rabbit: anti-CHMP4B (Proteintech; Cat. 13683-1-AP; batch 00019545; 1:250 for IF); anti-HD-PTP (Proteintech; Cat. 10472-1-AP; batch 00018154; 1:5000 for WB; 1:50 for IF) ( ); anti-UBAP1 (Proteintech; Cat. 12385-1-AP; batch 00014891) ( ); anti-EEA1 (Cell Signaling Technologies; C45B10; Cat. 3288S; batch 8; 1:400 for IF); anti-Rabex-5 (Thermo Scientific - Invitrogen; Cat. PA5-21117; batch RF2221849; 1:2500 for WB); anti-Rabaptin-5 (Proteintech; Cat. 14350-1-AP; batch 00005401; 1:5000 for WB); anti-Rab5 clone C8B1 (Cell Signaling Technologies; Cat. 3547S; batch 7; 1:500 for IF; 1:2500 for WB); anti-GAPVD1 (Abcam; Cat. ab86033; batch GR265707-8; 1:5000 for WB); anti-VPS4B (Proteintech; Cat. 17673-1-AP; batch 00009255; 1:2500 for WB); anti-exportin-1 (Bethyl Laboratories; Cat. A300-469A; batch 2).

Techniques: Activity Assay, Transfection, Fluorescence

Journal: Journal of Cell Science

Article Title: His domain protein tyrosine phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation

doi: 10.1242/jcs.259192

Figure Lengend Snippet: Model of the coupling of endosomal sorting and maturation. (i) HD-PTP initiates ESCRT-dependent MVB sorting of cargo by binding STAM2/ESCRT-0 while Rabex-5–Rabaptin-5 engages ubiquitylated cargo and is active to maintain high GTP-Rab5. (ii,iii) As cargo sorting proceeds, HD-PTP releases STAM2/ESCRT-0 and then binds CHMP4/ESCRT-III, while cargo is deubiquitylated. (iv) Cargo enters ILVs as CHMP4B/ESCRT-III is released. (v) HD-PTP, now free from ESCRTs, bind to Rabaptin-5. Rabex-5–Rabaptin-5 is inhibited, thus lowering GTP-Rab5 and promoting Rab conversion.

Article Snippet: Rabbit: anti-CHMP4B (Proteintech; Cat. 13683-1-AP; batch 00019545; 1:250 for IF); anti-HD-PTP (Proteintech; Cat. 10472-1-AP; batch 00018154; 1:5000 for WB; 1:50 for IF) ( ); anti-UBAP1 (Proteintech; Cat. 12385-1-AP; batch 00014891) ( ); anti-EEA1 (Cell Signaling Technologies; C45B10; Cat. 3288S; batch 8; 1:400 for IF); anti-Rabex-5 (Thermo Scientific - Invitrogen; Cat. PA5-21117; batch RF2221849; 1:2500 for WB); anti-Rabaptin-5 (Proteintech; Cat. 14350-1-AP; batch 00005401; 1:5000 for WB); anti-Rab5 clone C8B1 (Cell Signaling Technologies; Cat. 3547S; batch 7; 1:500 for IF; 1:2500 for WB); anti-GAPVD1 (Abcam; Cat. ab86033; batch GR265707-8; 1:5000 for WB); anti-VPS4B (Proteintech; Cat. 17673-1-AP; batch 00009255; 1:2500 for WB); anti-exportin-1 (Bethyl Laboratories; Cat. A300-469A; batch 2).

Techniques: Binding Assay